Ubiquitination, the covalent attachment of the small protein ubiquitin to other proteins, regulates a host of cellular processes. Protein ubiquitination has become a synonym for protein degradation. However, we are beginning to appreciate that a number of proteins are regulated by ubiquitination in a proteolysis-independent manner. Most of the current research is focused on the role of ubiquitin in targeting proteins for degradation by the 26S proteasome. However, proteome-wide approaches indicate that many proteins are regulated by proteolysis- independent ubiquitination and a detailed understanding of these processes is clearly important for biomedical research. Some of the key questions are: Why are some ubiquitinated proteins degraded and others are not? How can ubiquitination directly affect protein activity? Do proteins that are regulated in a proteolysis-independent manner by ubiquitin share sequence domains? In this proposal we use the yeast transcription factor Met4 as a model to study the mechanism of regulation of protein activity by proteolysis-independent ubiquitination (aim1);the role of ubiquitin-binding domains in protecting ubiquitinated proteins from degradation by the 26S proteasome and the role of ubiquitin-binding domains in regulating ubiquitin chain length (aim 2);and the physiological benefit of regulating protein activity by ubiquitination without degradation (aim 3). In aim 4 we use a proteome-wide approach to identify proteins that are regulated by proteolysis-independent ubiquitination and apply a bioinformatics strategy to identify shared protein domains. Ubiquitination affects many important cellular processes and has been linked to a number of human diseases including cancer, neurodegeneration, and retroviral infection. A contribution of proteolysis-independent ubiquitination in these diseases is emerging and it will be important to understand the mechanism behind this regulation to design diagnostic tools and treatment strategies. This proposal aims to achieve detailed mechanistic insight into proteolysis- independent regulation by ubiquitin and to define some of the characteristics of this regulatory ubiquitination pathway.